Duffy et al. BMC Biology (2020) 18:112 https://doi.org/10.1186/s12915-020-00803-6

REVIEW Open Access Modified nucleic acids: replication, evolution, and next-generation therapeutics Karen Duffy†, Sebastian Arangundy-Franklin† and Philipp Holliger*

Abstract Modified nucleic acids, also called xeno nucleic acids (XNAs), offer a variety of advantages for biotechnological applications and address some of the limitations of first-generation therapeutics. Indeed, several therapeutics based on modified nucleic acids have recently been approved and many more are under clinical evaluation. XNAs can provide increased biostability and furthermore are now increasingly amenable to in vitro evolution, accelerating lead discovery. Here, we review the most recent discoveries in this dynamic field with a focus on progress in the enzymatic replication and functional exploration of XNAs.

Nucleic acids: natural and expanded functions afford completely new modalities for therapeutic inter- A heritable genetic system is a defining requirement for vention (e.g., direct protein expression or specific and life. The natural nucleic acids, DNA and RNA, are ex- programmable modulation of gene expression) that can- quisitely suited to store and propagate genetic informa- not be easily accessed by other biologics or small mol- tion with sufficient stability and fidelity to support large ecule drugs. However, several challenges have thus far genomes but also the flexibility to enable evolution. Nu- prevented nucleic acids from reaching their full potential cleic acids are unique among biopolymers in that both as medicines including poor chemical and biological accessible information and functional capacity coexist in stability and narrow chemical diversity. a single molecule. Thus, function can be evolved at the To address these limitations, DNA and RNA analogues molecular level yielding nucleic acid-based ligands and have been developed and evaluated for their ability to . In addition, nucleic acids fold through predict- serve as useful biomaterials or functional molecules, en- able and (to a large extent) programmable interactions, code genetic information, and support evolution. These are highly water-soluble, and can be easily denatured synthetic genetic , broadly termed xeno nucleic and refolded. These advantages underpin their manifold acids (XNAs), exhibit modified backbones, sugars, or uses in biotechnology, diagnostics, therapeutics, nano- , and even novel bases or base pairs [10, 11]. technology, material science, , and data While natural nucleic acids may also be modified to storage. modulate their function in vivo (e.g., post-synthetic epi- The last four decades have seen the rise of nucleic genetic modifications) [12–17], we constrain ourselves acids as therapeutics [1], primarily in the form of anti- here to a discussion of nucleic acid congeners not found sense (ASOs) [2], small interfering in nature. We do, however, consider certain modifica- (siRNAs) [3], [4–6], [7], tions that occur sporadically in natural oligonucleotides, mRNAs [8], and gene-editing guides [9]. Nucleic acids such as 2′OMe or C5 modifications, to be XNAs in the case of fully (or heavily) substituted oligo- * Correspondence: [email protected] , the likes of which are not found in biology. †Karen Duffy and Sebastian Arangundy-Franklin contributed equally to this Several new and prominent XNA chemistries are shown work. in Fig. 1. One attractive feature of XNAs is their gener- MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK ally improved chemical and biological stability [18–20].

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Duffy et al. BMC Biology (2020) 18:112 Page 2 of 14

Fig. 1 (See legend on next page.) Duffy et al. BMC Biology (2020) 18:112 Page 3 of 14

(See figure on previous page.) Fig. 1 The chemical diversity of XNAs. XNAs are often categorized by the component of the (sugar, backbone, or base) carrying a modification. Shown here are XNAs discussed in this review, including both those of medical and historical relevance as well as several newly described chemistries. 2′F, 2′-fluoro; 2′OMe, 2′-O-methyl; LNA, ; FANA, 2′-fluoro arabinose nucleic acid; HNA, hexitol nucleic acid; 2′MOE, 2′-O-methoxyethyl; ribuloNA, (1′-3′)-β-L-ribulo nucleic acid; TNA, α-L-; tPhoNA, 3′-2′ phosphonomethyl-threosyl nucleic acid; dXNA, 2′-deoxyxylonucleic acid; PS, phosphorothioate; phNA, alkyl phosphonate nucleic acid; PNA, nucleic acid

Decoration with diverse chemical substituents (e.g., demonstration of genetic function in an uncharged back- hydrophobic groups) can also yield improved properties bone chemistry for the first time as well as in vitro evo- and functionalities such as new structural motifs and en- lution of phNA aptamers [29]. hanced target binding [21–24]. While a wide range of As certain individual modifications become easier to XNAs have been synthesized at the chemical level, synthesize and their unique properties better under- representing a rich palette to draw from, their functional stood, combining two or more of these known modifica- potential and usage is only just beginning to be probed. tions to sugars, backbones, and bases will further push Here, we explore recent discoveries in the enzymatic the boundaries of chemical diversity in XNAs [30–35]. synthesis and functional exploration of XNAs with a The development of ever more diverse chemistries view towards therapeutic applications. raises the question: how much of what we know about natural nucleic acids translates to XNA? To what extent Expanding the chemistry of nucleic acids can we borrow design strategies from natural nucleic Advances in nucleic acid chemistry have enabled the de- acids in order to rapidly develop useful new XNA chem- velopment of XNAs with improved base-pairing stability istries? Some design rules, such as the ability of certain over natural nucleic acids as well as enhanced activity in sequence motifs to form G-quadruplexes, have been the context of living tissues, leading to several FDA ap- shown to translate to TNA [36] and FANA [37]. How- proved nucleic acid therapeutics [25]. Research con- ever, the generality of such parallels remains to be explored. tinues apace to identify novel chemistries with increased potency, bioavailability, stability, decreased toxicity, and Templated synthesis of non-natural nucleic acids minimal off-target effects. The phosphoramidite approach to solid-phase DNA syn- An interesting development in this context is the dis- thesis [38, 39] developed in the early 1980s led to the main- covery that mixed backbone chemistries can display stream use of synthetic DNA in a myriad of applications. novel emergent properties. For example, Krishnamurthy Similarly, improvements in the synthesis of XNAs can be and colleagues probed the ability of XNA-RNA chimeric expected to increase their usage and unlock new applica- oligonucleotides to hybridize with their natural nucleic tions in the coming decades. XNA synthesis can be broadly acid counterparts and found that the otherwise non- divided into enzymatic and non-enzymatic approaches. pairing ribuloNA can be interspersed with RNA or DNA Some widely used XNAs (e.g., 2′OMe, LNA, PS, 2′MOE) building blocks to yield oligonucleotides with compar- can be chemically synthesized, although yields of even the able or higher duplex thermal stability than natural sys- most synthetically accessible XNAs are limited to around tems and tuneable base-pairing properties [26]. The 150 bp [40]. For other XNA chemistries, no reliable solid- authors suggest that such a system could be used to phase synthetic route is known [41]. Furthermore, solid- mimic natural base-pairing rules with a reduced number phase synthesis depends on explicit knowledge of the se- of bases, storing information in the sugar in place of the quence being synthesized. In contrast, templated synthesis GC . enables general information transfer and is essential for the Similarly, the recent discovery that smaller “skinny” evolution of functional oligonucleotides. (pyrimidine-like) and larger “fat” (-like) base pairs can form stable helices challenges conventional assump- Non-enzymatic templated synthesis tions of nucleic acid structure [27]. While the novel base Non-enzymatic templated synthesis traditionally uses acti- pairs did not lend stability when interspersed in a nat- vated nucleotides or short building blocks ural oligonucleotide duplex, fully skinny or fat duplexes that self-organize on a template via base-paring interac- showed increased thermal stability over the correspond- tions and react to polymerize. Such template copying, pio- ing natural duplexes. neered in the 1970s [42–44], has been extended to several In another example of XNAs reshaping classical as- XNA chemistries [45–53], often in the context of prebiotic sumptions [28], enzymatic synthesis of phosphonate nu- nucleic acid replication. For example, systems for tem- cleic acid (phNA), in which the negatively charged non- plated replication of PNA pentamers [54, 55] using reduc- bridging oxygen of the phosphodiester linkage is re- tive amination are efficient enough to permit model placed by an uncharged alkyl group, has enabled the selection experiments [56]. Similar strategies have also Duffy et al. BMC Biology (2020) 18:112 Page 4 of 14

been exploited to assemble nucleic acids via copper- a view to supersede phosphoramidite technology. Similarly, catalyzed click ligation of oligonucleotides [57, 58]andby enzymatic XNA synthesis may facilitate production of phosphoramidate ligation [59], and the resulting back- XNA oligonucleotides. XNAs with chemically conservative bones have shown some biocompatibility [60]. In an inter- modifications are accepted as substrates by natural poly- esting extension of this work, nucleic acid templates have merases, while engineering of to accept a also been used to organize chemical entities by proximity broader range of XNA substrates has also met with success for programmed reactions [61–63]. In particular, the Liu [72, 73]. Indeed, some polymerases have proven highly group has extended such templated synthesis approaches adaptable to new substrates. Thermophilic B-family poly- to chemically ligate diverse non-nucleic acid entities in merases, especially those from Pyrococcus and Thermococ- precise sequences defined by a DNA template [64]. Such cus genera, have proven especially amenable to engineering templated chemical syntheses allow increasingly divergent for XNA substrates [33, 74–81]. This functional plasticity chemistries to be synthesized and replicated without con- may be aided by high thermostability, which promotes straining diversity by the ability of its constituent greater tolerance for mutations that would otherwise be ex- monomers to serve as substrates for polymerases or other cessively destabilizing. However, A-family [82–90]and nucleic acid-modifying enzymes. mesophilic polymerases such as Phi29 [91, 92]aswellas RNA polymerases [93–97] have also been engineered to Enzymatic ligation and modification of XNAs accept XNA substrates with success (Table 1). However, Several DNA ligases have been shown to accept non- the efficiency, fidelity, and kinetics of engineered polymer- natural substrates and have been used alone or in con- ases on XNA substrates are usually compromised relative junction with XNA polymerases to synthesize XNA oli- to the native enzymes and natural substrates. Often, “for- gonucleotides. Much like the aforementioned chemical cing” conditions (e.g., superstoichiometric con- ligation strategies, enzymatic ligation of pre-organized centrations or the presence of Mn2+, which in turn reduces on a template allows for positioning of modi- fidelity) are needed to boost synthetic yields [106]. fied nucleotides, including multiple different modifica- tions, at defined loci. It has recently been shown that Capitalizing on the promiscuity of natural polymerases several commercially available ligases can catalyze It is perhaps remarkable that natural polymerases can ligation of XNA substrates including 2′OMe, HNA, incorporate modified nucleotides at all, given their need LNA, TNA, and FANA [41, 65]. Moreover, rational for stringent substrate specificity and accuracy. However, design and molecular modeling approaches have now at some positions in the nucleotide, modifications are resulted in the first XNA-templated XNA ligase [66]. readily tolerated. For example, modifications at the C5 The Liu and Hili groups have also worked extensively position of and C7 position of N7-deaza- with ligase-mediated synthesis of modified DNA from project into the duplex’s major groove and do diversely functionalized DNA 3- or 5-mers [67, 68], not interfere with Watson-Crick base pairing. Therefore, allowing a great variety of modifications, including even bulky adducts at these positions are generally well hydrophobic, aliphatic, aromatic, acid, and basic moi- tolerated by polymerases [22, 107–112]. Enzymatic syn- eties, to be incorporated using these short “quasico- thesis of nucleotides with reactive groups (e.g., for dons.” In agreement with results obtained with copper-catalyzed click chemistry) at these positions can SOMAmer (Slow Off-rate Modified ) selections allow elaborate post-synthetic modifications [113–117]. [22], they find that functionalization with nonpolar moi- The plasticity of polymerases has also allowed researchers eties appears to promote faster selection convergence to carry out wholesale replacement of natural bases with and stronger aptamer binding [21]. Incorporating chem- close chemical analogues [118, 119]. In some cases, synthe- ical diversity beyond that found in proteins (e.g., haloge- sis is efficient enough to produce modified PCR products of nated residues), the group isolated high affinity aptamers up to 1.5 kb [119]. Moreover, entirely new unnatural base against PCSK9 and interleukin-6 [69]. The above strategies pairs (UBPs) have been developed based on novel encapsulate the paradigm of leveraging the unique proper- bonding patterns [105, 120], hydrophobicity [121, 122], and ties of nucleic acids (i.e., encoded synthesis, evolvability) shape complementarity [123–125]. These UBPs can be rep- coupled with an expanded set of chemical substituents to licated by engineered and natural polymerases, demonstrat- create functional molecules with therapeutic potential. ing successful expansion of the genetic code, and recently achieving information encoding in an unprecedented eight- Polymerase synthesis of XNAs letter genetic alphabet [120]. Enzymatic polymerization of nucleic acids is orders of magnitude faster and more accurate than chemical copy- XNA synthesis in vivo ing or synthesis and is now being pursued for next- The synthesis and replication of XNAs in vivo represents generation DNA oligonucleotide synthesis [70, 71], with an important frontier in XNA research towards aims Duffy et al. BMC Biology (2020) 18:112 Page 5 of 14

Table 1 Polymerase-mediated synthesis of XNAs Pol Family Polymerase Novel Activity Ref. Pol A Taq 2’F RNA [83, 84, 87, 88, 90, 98] Tth 2’OME RNA Pol θ 2’-azido RNA

Pol B Tgo CeNA [29, 33, 75, 78, 81, 82, 92] KOD LNA 9°N phNA Pfu HNA phi29 FANA CyDNA 2’F RNA ANA TNA 2’azido RNA tPhoNA Pol Y (D-aa) Dpo4 L-DNA [99–101]

Pol X (D-aa) ASFV pol L-DNA [102] L-RNA

RNAP T7 RNAP 2’F RNA [95, 97, 103, 104] Syn5 2’OMe RNA Ds-Pa UBP

RT HIV-RT pyDAD-puADA UBP [105]

Natural and engineered polymerases across of wide range of families have been used to synthesize unnatural nucleic acids. Polymerases are shown as space- filling models with primer shown in blue and templates shown in red. PDB codes are 4BWJ (Taq), 5OMF (KOD), 4G3I (Dpo4), 5HRF (ASFV), 1H38 (T7 RNAP), and 6HAK (HIV RT) Duffy et al. BMC Biology (2020) 18:112 Page 6 of 14

such as stable encoding of unnatural amino acids and temperatures for evolution of non-thermostable enzymes genetic orthogonality for “firewalling” synthetic biology. [91]. Compartmentalized self-tagging (CST) [144] allows To this end, complete replacement of and/or selection for much more difficult substrates or condi- with 5-substituted pyrimidine analogues in tions; selection is based on a primer extension templated bacterial genomes has now been achieved by careful by the encoding . The biotinylated primer along metabolic engineering and evolution [126–129]. with any that have been “captured” by suffi- Remarkably, Romesberg and colleagues have been able cient primer extension are partitioned on streptavidin to engineer E. coli strains with the capacity to replicate beads. CST has yielded polymerases for a range of XNAs and maintain their unnatural NaM–TPT3 base pair in [78], most recently for dxNA [145]. In contrast to bulk both plasmid [130] and genomic [131] contexts. To emulsification methods, microfluidic devices have been achieve this, they engineered a triphosphate used to generate highly homogeneous emulsion droplets, transporter [132], invoked Cas9 to degrade sequences enhancing the ability to accurately distinguish small in- having lost the UBP, and made several tweaks to the E. cremental improvements important in stepwise selec- coli DNA synthesis and repair machinery. They further tions [146, 147]. Another directed evolution strategy is demonstrated the compatibility of the UBP with transla- phage display, whereby polymerases displayed on phage tion machinery to site-specifically encode unnatural particles are challenged to extend a primer with separ- amino acids [133, 134]. Further semi-synthetic organisms able (e.g., biotinylated) nucleotides [80, 89, 148]. In a with substituted genomes or the ability to propagate UBPs notable recent use of phage display for polymerase evo- will likely follow these early successes [135, 136]. lution, Chen et al. identified several Taq variants capable of synthesizing and reverse transcribing 2′OMe RNA Directed evolution for XNA polymerase engineering [88], an XNA of particular interest for therapeutics due While base modifications and even completely new base to its high biostability and resistance. pairs are tolerated by natural polymerases to varying ex- tents, modifications to the sugar and backbone generally Rational engineering of XNA polymerases: structural prove more challenging, especially at full substitution. insights To improve activity with such unnatural substrates, Polymerases make many key contacts with the incoming polymerase engineering approaches, ranging from ra- nucleotide triphosphate and primer strand during the tional engineering driven by structural insights or com- catalytic cycle [149]. Structures of polymerases in com- putational analysis to screening and directed evolution, plex with modified substrates may therefore aid in both are often employed. Among these, emulsion-based rational engineering and retrospective understanding of methods and phage display have been especially useful XNA polymerases. Recently, Kropp et al. solved ternary [137, 138]. structures of KlenTaq polymerase with a C5-modified A range of directed evolution strategies have been cytidine at each of six successive positions [150] in the employed in the field. Compartmentalized self- catalysis cycle, recapitulating the movement of the modi- replication (CSR) [139–141] challenges polymerases to fied substrate through the polymerase’s and PCR amplify their own gene inside an emulsion com- revealing a surprising level of flexibility in both the partment. The exponential enrichment of highly active modified nucleotide and the interacting polymerase resi- clones, as opposed to simple partitioning of active vari- dues to accommodate the modification. ants, makes CSR a powerful method. However, it places Further structural insight into XNA polymerization stringent demands on polymerase performance, which has been reported by Singh et al. [151] with structures are somewhat relieved in short-patch CSR (spCSR) [85] of an engineered KlenTaq in a binary pre-incorporation where amplification is confined to only a short section and a ternary post-incorporation complex with the un- of the polymerase gene. Nested CSR allows selection of natural base pair dZ:dP. Although none of the mutated sequences or features not present in the polymerase amino acids are in direct contact with primer, template, gene itself (e.g., novel base pairs, reverse of or incoming dZTP, the structure suggests that increased XNA templates) by using out-nesting primers with these flexibility, particularly in the thumb subdomain, and an features during the CSR amplification step [79, 86]. A increased angle of closure in the finger subdomain facili- further extension called compartmentalized partnered tate primer elongation with the unnatural base pair. replication (CPR) can be used to select for enzymatic Chim et al. recently reported ternary structures of a activities other than nucleic acid polymerization by con- hyperthermophilic B-family polymerase, an engineered straining the selectable PCR reaction by the fitness of a KOD mutant, polymerizing TNA [152]. Given the preva- partner gene [86, 142]. Recently, CSR has also been lent use of B-family hyperthermophiles in polymerase adapted for isothermal amplification reactions (iCSR), engineering, the structures of the apo, binary, and tern- both at high temperatures [143] and at lower ary polymerase complexes will prove useful in Duffy et al. BMC Biology (2020) 18:112 Page 7 of 14

developing hypotheses for further work in the field. Sub- RNA polymerases have also been engineered to sequently published ternary structures of KOD and 9°N synthesize XNAs. It has been shown that T7 RNAP can with DNA primer and template and an incoming dNTP transcribe content-expanded RNA from DNA containing [149, 153] suggest possible explanations for the predis- the Ds-Pa UPB as well as further modified Pa bases position of hyperthermophilic archaeal B-family poly- [103]. Through a small screen of previously identified merases to engineering: an unusually wide and positively mutations, Kimoto et al. identified a T7 RNAP mutant charged channel between the finger and thumb subdo- that incorporates 2′-F and cytidine triphosphates mains may allow space for substrate modifications while and shows improved incorporation of Pa nucleotides maintaining strong binding to the template and high and their analogues in difficult sequence contexts [93]. processivity. Additionally, whereas the primer template Surprisingly, several bulky modifications of the Pa nu- duplex adopts an A-form in the active site of A-family cleotide designed to expand chemical diversity for apta- polymerases, it adopts a B-form in KOD, possibly con- mer and selections appear to be even better tributing to accommodation of nucleotide modifications substrates than the original Pa triphosphates. Similarly, a in the B-form duplex’s wider major groove. Tgo polymerase mutant previously evolved to synthesize HNA was recently shown to incorporate HNA nucleo- Rational engineering of XNA polymerases: translation of tides with various aromatic modifications on the mutations across substrates and polymerases base [35]. Here too, some of the bulkier base modifica- There are numerous examples of transferable mutations tions demonstrated superior incorporation. across XNA substrates and polymerases. A recent report [154] details engineering of a previously described Taq Reverse transcription of XNAs mutant to better incorporate 2′ modified XNAs. From Identification of enzymes capable of reverse transcribing kinetic data on incorporation of different 2′ modified XNAs demonstrates the potential of these divergent nucleotides, relevant mutations from the literature were chemistries as genetic materials and is crucial for chosen based on the hypothesis that the rate-limiting in vitro selections [156]. Some XNAs can be reverse step may be recognition of the modified primer strand. transcribed by natural polymerases (e.g., TNA and Several mutants showed enhanced synthesis of 2′F and FANA by Bst polymerase [157–159]). Alternately, engin- 2′OMe and/or reverse transcription of 2′OMe RNA. In eering approaches must be taken [79, 88]. A small num- another instance, diversification at eight “specificity de- ber of mutations in Tgo polymerase yielded a reverse termining residues” selected by computational analysis, transcriptase with a generally expanded substrate evolutionary conservation, and literature precedent spectrum that can reverse transcribe several XNAs to yielded improved polymerases for RNA and TNA [75]. DNA [29, 78]. Furthermore, testing the top mutation sets in the con- text of different homologous B-family polymerases evi- Analysis of XNAs and XNA polymerases denced their general utility as well as revealing the One difficulty in working with XNAs is that they are structural context in which they functioned best. Simi- often incompatible with traditional methods of nucleic larly, polymerase synthesis of increasingly diverse XNAs acid manipulation or analysis such as restriction en- was achieved with a TNA polymerase by combining the zymes and sequencing technologies. Thus, an important TNA sugar with base modifications known to be parallel effort to the development of XNAs and XNA polymerase-compatible [31, 32]. While it has been gen- polymerases is the development of tools to analyze them. erally believed that mutation of conserved residues is For example, fidelity has not been evaluated in detail for highly deleterious, it is becoming increasingly apparent many of the described XNA polymerases. A recent through this and other work that such mutations can be key method for assaying fidelity reported finding that even in allowing activity with unnatural substrates [75, 155]. relatively small and natural RNA modifications can sig- Liu et al. recently reported engineering of a polymer- nificantly increase polymerase error rates [160]. Deep se- ase to synthesize a newly described XNA, tPhoNA, with quencing has also been used to investigate error profiles both sugar and backbone modifications [33]. Impres- and polymerase read-through of templates with sively, the polymerase engineering strategy consisted backbone modifications [161] and found that some com- solely of successive introduction and evaluation of muta- monly used isosteric modifications, such as phosphor- tions at positions known to affect synthesis for other othioates, caused significant copying errors. This fidelity XNA substrates. Their success is a testament to the ac- data foreshadows an important limitation in enzymatic cumulating knowledge base in the field of polymerase XNA synthesis that will likely need to be addressed as engineering and the extraordinary ability of a small the field moves forward. On the other hand, the promis- number of specific key mutations to confer an expanded cuous behavior of polymerases has been leveraged to substrate spectrum. read and record the presence of epigenetic DNA and Duffy et al. BMC Biology (2020) 18:112 Page 8 of 14

RNA modifications via misincorporation “signatures” at Pegaptanib, while discovered as an RNA aptamer, under- the modifications [162–170]. In addition, the first in- went rounds of medicinal chemistry-like optimization stance of direct sequencing of an XNA was recently re- and modification with 2′OMe and 2′F moieties to in- ported; FANA was sequenced using nanopore crease its in vivo stability and potency. Over 100 oligo- technology, albeit with relatively short read lengths nucleotide drugs are currently in clinical trials [185], [171]. evidencing the belief and momentum behind these new An XNA particularly limited by a lack of tools is L- therapeutic modalities able to address previously DNA. Such “mirror image” DNA provides both undruggable target classes. complete orthogonality in macromolecule-scale features Somalogic has pioneered aptamer discovery with their and interactions, while maintaining identical properties SOMAmer platform [186], which employs modifications to DNA at the chemical level. Thus, enzymatic synthesis at the C5 position of pyrimidine nucleotides to append of L-DNA requires mirror image polymerases made of protein-like side chains to aptamer libraries, generating D-amino acids. Zhu and colleagues first made the D- aptamers of high affinity and specificity for diagnostic form of the smallest known polymerase, African Swine and clinical applications. Fever Virus polymerase X, and used it to synthesize L- As mentioned above, aptamers modified with 2′F and DNA, showing that the polymerase was strictly enantio- 2′OMe moieties have been selected previously, but Liu specific with no crossover inhibition from D-nucleotide and colleagues have recently made possible direct selec- triphosphates [102]. The group subsequently reported tions in the latter backbone at full substitution using two synthesis of a mutant of the larger thermostable Dpo4 Taq polymerase variants [104]. Similarly, direct selec- polymerase with D-amino acids, which enabled PCR tions of TNA have recently yielded aptamers against amplification of an L-DNA product [99, 100]. Kluss- proteins [187] and small molecule targets [188]. Pursu- mann and colleagues had also reported synthesis of a D- ant to the hypothesis that UBPs increase the sequence Dpo4 mutant [101], which was used to assemble gene- space and thus functional diversity of nucleic acid se- length L-DNA sequences. A mirror image ligase has also quences, aptamers containing UBPs have been selected been reported [172]. Mirror image nucleic acids are of against both protein targets [189–191] and whole cells immense interest for therapeutics and are being actively [192, 193]. pursued in research and clinical development [173]. In many cases, aptamers are selected as DNA or RNA However, their usage remains limited by the arduous sequences and then modified post-selection. Commonly, process of synthesizing D-polymerases and the incom- DNA and RNA aptamers are converted to their 2′ modi- patibility of L-DNA with traditional nucleic acid ma- fied counterparts, producing useful affinity reagents for nipulation tools. applications that would be incompatible with natural nu- cleic acids (e.g., for stability in alkaline conditions) [194]. Non-natural nucleic acids for therapeutic In many cases, such post-SELEX modifications decrease applications affinity of the original aptamer, arguing for direct selec- Current FDA- and EMA-approved nucleic acid thera- tion in XNA systems where possible. However, this is peutics (Table 2) can be broadly defined in three cat- not always the case. A single base modification intro- egories: antisense (ASO), aptamer, and most recently, duced in a thrombin aptamer increased its binding affin- siRNA. Given the inherent instability of natural nucleic ity by up to 7-fold [195]. In another recent report, acids in biological fluids, it is not surprising that every Sullenger and colleagues modified RNA aptamers example from these categories is either fully or partly against prostate cancer cells with 2′F pyrimidines. Upon modified. Fomivirsen, a PS ASO to treat cytomegalovirus conjugation with small molecule drugs or therapeutic retinitis (CMV), was the first nucleic acid therapeutic to , these aptamers displayed significant specificity gain regulatory approval. It was subsequently withdrawn and toxicity against cancerous but not normal cells [196, from the market due to a lack of demand as incidence of 197]. We direct the reader to a recent review for a more (CMV) infections decreased sharply [184]. Nevertheless, detailed panorama of modified aptamer chemistry and it proved an important step in demonstrating the safety related technologies [198]. and potential of nucleic acid medicines. Eteplirsen and XNAs are also being explored for other new thera- Golodirsen are fully composed of a phos- peutic modalities beyond siRNA, ASOs, and aptamers. phoramidate (PMO) backbone, while Mipomersen, Modified oligonucleotides capable of cleaving or ligating Nusinersen, Inotersen, and Volanesorsen feature both other oligonucleotides have been developed as fully phosphorothioate (PS) and 2′methoxyethyl (2′MOE) modified “XNAzymes” [156]. In other cases, incorpor- modifications. Patisiran contains 2′OMe pyrimidine resi- ation of modified nucleotides at specific positions can dues in both siRNA strands. The siRNA Givosiran also expand chemical functionality or otherwise enhance ca- carries PS linkages and 2′F and 2′OMe modifications. talysis [199, 200]. Therapeutic XNAs are also being Duffy et al. BMC Biology (2020) 18:112 Page 9 of 14

Table 2 FDA-approved nucleic acid therapeutics as of February 2020 Drug name Target Modifications Mechanism Indication Approval Ref. (trade name) Fomivirsen mRNA of the CMV PS ASO Cytomegalovirus retinitis FDA (1998) and EMA (1999) approved. [174] (Vitravene) immediate-early (IE)-2 (translation (CMV) FDA (2001) and EMA (2002) withdrawn protein blocking) Pegaptanib Vascular endothelial 2′F, 2′OMe, PEG Aptamer Neovascular (wet) age- FDA approved (2004) [175] (Macugen) growth factor (VEGF165) conjugate related macular degeneration Mipomersen Apolipoprotein B-100 2′MOE, PS, 5mC ASO (RNase Homozygous familial FDA approved (2013) [176] (Kynamro) mRNA H) hypercholesterolemia Eteplirsen Exon 51 in dystrophin PMO ASO Duchenne muscular FDA approved (2016) [177] (Exondys 51) mRNA (splicing dystrophy modulation) Nusinersen Survival of motor 2′MOE, PS, 5mC ASO Spinal muscular atrophy FDA (2016) and EMA (2017) approved [178] (Spinraza) neuron 2 (SMN2) pre- (splicing mRNA modulation) Patisiran Transthyretin (TTR) 2′OMe siRNA Hereditary transthyretin- FDA and EMA approved (2018) [179] (Onpattro) mRNA mediated amyloidosis Inotersen Transthyretin (TTR) 2′MOE, PS, 5mC ASO (RNase Hereditary transthyretin- FDA and EMA approved (2018) [180] (Tegsedi) mRNA H) mediated amyloidosis

Volanesorsen Apolipoprotein C3 (apo- 2′MOE, PS, 5mC ASO (RNase Familial chylomicronemia EMA approved (2019) [181] (Waylivra) CIII) mRNA H) syndrome Givosiran Aminolevulinate PS, 2′F, 2′OMe, siRNA Acute hepatic porphyria FDA approved (2019) [182] (Givlaari) synthase 1 (ALAS1) GalNAc mRNA conjugate Golodirsen Exon 53 in dystrophin PMO ASO Duchenne muscular FDA approved (2019) [2, (Vyondys 53) mRNA (splicing dystrophy 183] modulation) explored in the context of mRNAs [201], microRNAs steep increase in clinical-stage nucleic acid medicines in [202], anti-gene oligonucleotides [203], and CRISPR the coming decades. guides [204, 205]. Authors’ contributions Delivery of oligonucleotides into the cytosol of target The manuscript was written and revised jointly by K.D. S.A.-F., and P.H. All cells remains one of the major challengers in the devel- authors reviewed and approved the manuscript. opment of nucleic acid therapeutics [206, 207]. Conjuga- Funding tion or encapsulation in lipids has demonstrated This work was supported by a Gates Cambridge Scholarship (K.D.), a research promise in improving pharmacokinetics and biodistribu- collaboration between AstraZeneca UK Limited and the Medical Research tion [208], and -penetrating peptides can increase Council- MRC-Astra Zeneca Blue Sky Grant (S.A-F.), and the Medical Research Council (program no. MC_U105178804) (P.H.). transport of biomolecules across cellular membranes [209, 210]. Other conjugations can enable targeting to Availability of data and materials specific tissues with high efficiency; N-acetylgalactosa- Not applicable. mine (GalNAc) has proven highly efficient for targeting Competing interests biomolecules to the liver [211]. However, on the whole, The authors declare that they have no competing interests. effective delivery is a major challenge that remains to be addressed by the field. References The future of unnatural nucleic acid therapeutics 1. Khvorova A, Watts JK. The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol. 2017;35(3):238–48. As tools such as XNA polymerases become available for 2. Shen X, Corey DR. Chemistry, mechanism and clinical status of antisense an increasing number and diversity of nucleic acid oligonucleotides and duplex RNAs. Nucleic Acids Res. 2018;46(4):1584–600. chemistries, it is becoming possible to explore in greater 3. Burnett JC, Rossi JJ. RNA-based therapeutics: current progress and future prospects. Chem Biol. 2012;19(1):60–71. detail the molecular properties and therapeutic potential 4. Maier KE, Levy M. From selection hits to clinical leads: progress in aptamer of these unnatural polymers. Several chemistries show discovery. Mol Ther Methods Clin Dev. 2016;5:16014. promise in addressing the shortcomings that limit the 5. Zhou J, Rossi J. Aptamers as targeted therapeutics: current potential and challenges. Nat Rev Drug Discov. 2017;16(3):181–202. utility of natural nucleic acids in therapeutic contexts. 6. Nimjee SM, White RR, Becker RC, Sullenger BA. Aptamers as therapeutics. We anticipate that exploration in this field will lead to a Annu Rev Pharmacol Toxicol. 2017;57:61–79. Duffy et al. BMC Biology (2020) 18:112 Page 10 of 14

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